UW-preservation of cultured human gallbladder epithelial cells: phenotypic alterations and differential mucin gene expression in the presence of bile.
Campion J P,Porchet N,Aubert J P,L'Helgoualc'h A,Clément B
Hepatology (Baltimore, Md.)
In orthotopic liver transplantation, extended cold ischemia of the graft may induce cell damage, particularly in biliary epithelium. We have investigated the effects of a cold University of Wisconsin (UW) solution on cultured human gallbladder biliary epithelial cells (GBEC) exposed or not exposed to stagnant bile. In UW solution, morphological alterations of cultured GBEC were not prominent under light microscopy after 16 hours at 4 degrees C, being more striking after 24 to 48 hours. Ultrastructural examination of GBEC showed a condensation of chromatin at the periphery of the nuclei after 16 hours in cold UW solution. Both protein and DNA syntheses were strikingly reduced in these cells. After rewarming in standard Williams' medium at 37 degrees C for 24 hours, cultured GBEC exhibited both normal morphology and function. As in both freshly isolated and routinely cultured GBEC, rewarmed cells expressed various mucin genes, namely MUC1, MUC3, MUC4, MUC5AC, and MUC5B genes, whereas MUC2 mRNAs were barely detectable. A dramatic decline in the steady-state mRNA levels of both MUC3 and MUC5B was found in cultured GBEC versus freshly isolated cells. Addition of bile into UW solution at 4 degrees C had no significant effect on GBEC morphology and DNA and protein syntheses. When bile was added during the rewarming period, both protein and DNA syntheses were strongly reduced. Addition of bile during either storage in UW solution or rewarming period induced increased steady-state MUC2, MUC3 and MUC5AC mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega-3 fatty acids.
El-Badry Ashraf Mohammad,Moritz Wolfgang,Contaldo Claudio,Tian Yinghua,Graf Rolf,Clavien Pierre-Alain
Hepatology (Baltimore, Md.)
UNLABELLED:Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega-6 (n-6): omega-3 (n-3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline-deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega-3 and omega-6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega-3 PUFAs and compared with the control diet-fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega-6: omega-3 PUFA ratio. Dietary omega-3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. CONCLUSION:Macrosteatotic livers disclosed an abnormal omega-6: omega-3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega-3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool.
10.1002/hep.21625
Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice.
Hepatology (Baltimore, Md.)
Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild-type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/-) were transplanted into wild-type recipient mice. Mdr2+/- mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2-/- mice, the Mdr2+/- mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild-type donors. Liver grafts from Mdr2+/- donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/- livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild-type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/- grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation.
10.1002/hep.21169
Effect of tauroursodeoxycholic acid on bile flow and calcium excretion in ischemia-reperfusion injury of rat livers.
Ono T,Nagasue N,Kohno H,Uchida M,Takemoto Y,Dhar D K,Nakamura T
Journal of hepatology
BACKGROUND/AIMS:Tauroursodeoxycholic acid is known to have a hepatoprotective action in cholestatic disorders. We evaluated whether oral pretreatment with tauroursodeoxycholic acid could protect the liver from ischemia-reperfusion injury, with particular regard to its effect on bile flow and biliary calcium excretion. METHODS:A 1-hour in vivo ischemia-reperfusion model of 70% of the lobes of rat liver was used. Animals were divided into six groups (each group; n = 8); a non-ischemia sham group (CS), a control group without bile acids (CON), and 4 bile acid groups; 10 mg/kg and 50 mg/kg (U10, U50), taurocholic acid 10 mg/kg (CA10) and tauroursodeoxycholic acid 10 mg/kg (CD10). Bile acids were given orally for 7 days before operation. RESULTS:Three hours after reperfusion, oral bile acid pretreatment failed to reduce the hepatic ischemia-reperfusion injury biochemically, but histological improvement was observed in the tauroursodeoxycholic acid groups. After reperfusion, tauroursodeoxycholic acid significantly increased bile flow from the ischemic liver, and also significantly increased serum calcium concentration. Although tauroursodeoxycholic acid did not change biliary calcium concentration, it significantly enhanced total biliary calcium output during reperfusion. CONCLUSION:Thus, tauroursodeoxycholic acid inhibited tissue calcium accumulation and enhanced sinusoidal and biliary calcium output during hepatic ischemia-reperfusion. However, it is still unclear if calcium mobilization is part of the protective mechanisms of tauroursodeoxycholic acid in ischemia-reperfusion injury of the liver.
Endothelin B receptor-mediated protection against anoxia-reoxygenation injury in perfused rat liver: nitric oxide-dependent and -independent mechanisms.
Taniai H,Suematsu M,Suzuki T,Norimizu S,Hori R,Ishimura Y,Nimura Y
Hepatology (Baltimore, Md.)
This study aimed to investigate the roles of endothelin (ET) receptors in biliary dysfunction and cell injury in postischemic livers. Rat livers perfused with oxygenated Krebs-Henseleit solution were exposed to reoxygenation following 20-minute hypoxia. The anoxic perfusion decreased bile output and reduced cyclic guanosine monophosphate (cGMP) contents, an index of nitric oxide (NO) generation. Upon reoxygenation, the decreased bile was not fully recovered, and the resistance increased biphasically: an early transient spike accompanied by an elevated release of ET-1 and a rise accompanied by a cGMP elevation in the later period. The initial vasoconstriction appeared to be mediated by both ET(A) and ET(B) receptors, as judged by inhibitory effects of their antagonists, BQ-485 and BQ-788, respectively, while the late elevation of the resistance was not attenuated by these reagents, but rather enhanced by the ET(B) blockade. The BQ-788 treatment attenuated the reoxygenation-induced cGMP elevation and induced bile acid-dependent choleresis. However, such a change upon the ET(B) blockade coincided with dissociation of a recovery of phospholipids and aggravation of cell injury. The BQ-788-elicited deterioration of reoxygenation-elicited changes was attenuated by NO supplement with S-nitroso-N-acetyl penicillamine. N(omega)-Nitro-L-arginine methyl ester, an NO synthase inhibitor, mimicked biliary changes elicited by the ET(B) blockade but without causing notable cell injury. Under these circumstances, coadministration of clotrimazole, an inhibitor of cytochrome P450 mono-oxygenases, elicited the injury comparable with that observed under the ET(B) blockade. These results suggest that ET(B)-mediated signaling limits excessive bile acid excretion and plays a protective role against reoxygenation injury through mechanisms involving both NO-dependent and -independent processes.
10.1053/jhep.2001.23001
Systemic protection through remote ischemic preconditioning is spread by platelet-dependent signaling in mice.
Oberkofler Christian E,Limani Perparim,Jang Jae-Hwi,Rickenbacher Andreas,Lehmann Kuno,Raptis Dimitri A,Ungethuem Udo,Tian Yinghua,Grabliauskaite Kamile,Humar Rok,Graf Rolf,Humar Bostjan,Clavien Pierre-Alain
Hepatology (Baltimore, Md.)
UNLABELLED:Remote ischemic preconditioning (RIPC), the repetitive transient mechanical obstruction of vessels at a limb remote to the operative site, is a novel strategy to mitigate distant organ injury associated with surgery. In the clinic, RIPC has demonstrated efficacy in protecting various organs against ischemia reperfusion (IR), but a common mechanism underlying the systemic protection has not been identified. Here, we reasoned that protection may rely on adaptive physiological responses toward local stress, as is incurred through RIPC. Standardized mouse models of partial hepatic IR and of RIPC to the femoral vascular bundle were applied. The roles of platelets, peripheral serotonin, and circulating vascular endothelial growth factor (Vegf) were studied in thrombocytopenic mice, Tph1(-) (/) (-) mice, and through neutralizing antibodies, respectively. Models of interleukin-10 (Il10) and matrix metalloproteinase 8 (Mmp8) deficiency were used to assess downstream effectors of organ protection. The protection against hepatic IR through RIPC was dependent on platelet-derived serotonin. Downstream of serotonin, systemic protection was spread through up-regulation of circulating Vegf. Both RIPC and serotonin-Vegf induced differential gene expression in target organs, with Il10 and Mmp8 displaying consistent up-regulation across all organs investigated. Concerted inhibition of both molecules abolished the protective effects of RIPC. RIPC was able to mitigate pancreatitis, indicating that it can protect beyond ischemic insults. CONCLUSIONS:We have identified a platelet-serotonin-Vegf-Il10/Mmp8 axis that mediates the protective effects of RIPC. The systemic action, the conservation of RIPC effects among mice and humans, and the protection beyond ischemic insults suggest that the platelet-dependent axis has evolved as a preemptive response to local stress, priming the body against impending harm.
10.1002/hep.27089
Antibiotic pretreatment alleviates liver transplant damage in mice and humans.
The Journal of clinical investigation
Although modifications of gut microbiota with antibiotics (Abx) influence mouse skin and cardiac allografts, its role in orthotopic liver transplantation (OLT) remains unknown. We aimed to determine whether and how recipient Abx pretreatment may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. Mice (C57BL/6) with or without Abx treatment (10 days) were transplanted with allogeneic (BALB/c) cold-stored (18 hours) livers, followed by liver and blood sampling (6 hours). We divided 264 human OLT recipients on the basis of duration of pre-OLT Abx treatment into control (Abx-free/Abx <10 days; n = 108) and Abx treatment (Abx ≥10days; n = 156) groups; OLT biopsy (Bx) samples were collected 2 hours after OLT (n = 52). Abx in mice mitigated IRI-stressed OLT (IRI-OLT), decreased CCAAT/enhancer-binding protein homologous protein (CHOP) (endoplasmic reticulum [ER] stress), enhanced LC3B (autophagy), and inhibited inflammation, whereas it increased serum prostaglandin E2 (PGE2) and hepatic PGE2 receptor 4 (EP4) expression. PGE2 increased EP4, suppressed CHOP, and induced autophagosome formation in hepatocyte cultures in an EP4-dependent manner. An EP4 antagonist restored CHOP, suppressed LC3B, and recreated IRI-OLT. Remarkably, human recipients of Abx treatment plus OLT (Abx-OLT), despite severe pretransplantation clinical acuity, had higher EP4 and LC3B levels but lower CHOP levels, which coincided with improved hepatocellular function (serum aspartate aminotransferase/serum aspartate aminotransferase [sALT/sAST]) and a decreased incidence of early allograft dysfunction (EAD). Multivariate analysis identified "Abx-free/Abx <10 days" as a predictive factor of EAD. This study documents the benefits of Abx pretreatment in liver transplant recipients, identifies ER stress and autophagy regulation by the PGE2/EP4 axis as a homeostatic underpinning, and points to the microbiome as a therapeutic target in OLT.
10.1172/JCI127550
Fasting protects liver from ischemic injury through Sirt1-mediated downregulation of circulating HMGB1 in mice.
Rickenbacher Andreas,Jang Jae Hwi,Limani Perparim,Ungethüm Udo,Lehmann Kuno,Oberkofler Christian E,Weber Achim,Graf Rolf,Humar Bostjan,Clavien Pierre-Alain
Journal of hepatology
BACKGROUND & AIMS:Fasting and calorie restriction are associated with a prolonged life span and an increased resistance to stress. The protective effects of fasting have been exploited for the mitigation of ischemic organ injury, yet the underlying mechanisms remain incompletely understood. Here, we investigated whether fasting protects liver against ischemia reperfusion (IR) through energy-preserving or anti-inflammatory mechanisms. METHODS:Fasted C57BL6 mice were subjected to partial hepatic IR. Injury was assessed by liver enzymes and histology. Raw264-7 macrophage-like cells were investigated in vitro. Sirt1 and HMGB1 were inhibited using Ex527 and neutralizing antibodies, respectively. RESULTS:Fasting for one, but not two or three days, protected from hepatic IR injury. None of the investigated energy parameters correlated with the protective effects. Instead, inflammatory responses were dampened in one-day-fasted mice and in starved macrophages. Fasting alone led to a reduction in circulating HMGB1 associated with cytoplasmic HMGB1 translocation, aggregate formation, and autophagy. Inhibition of autophagy re-elevated circulating HMGB1 and abolished protection in fasted mice, as did supplementation with HMGB1. In vitro, Sirt1 inhibition prevented HMGB1 translocation, leading to elevated HMGB1 in the supernatant. In vivo, Sirt1 inhibition abrogated the fasting-induced protection, but had no effect in the presence of neutralizing HMGB1 antibody. CONCLUSIONS:Fasting for one day protects from hepatic IR injury via Sirt1-dependent downregulation of circulating HMGB1. The reduction in serum HMGB1 appears to be mediated by its engagement in the autophagic response. These findings integrate Sirt1, HMGB1, and autophagy into a common framework that underlies the anti-inflammatory properties of short-term fasting.
10.1016/j.jhep.2014.04.010
Prevention of ischemia/reperfusion injury in the rat liver by atrial natriuretic peptide.
Bilzer M,Witthaut R,Paumgartner G,Gerbes A L
Gastroenterology
BACKGROUND/AIMS:Atrial natriuretic peptide (ANP) protects against hypoxia/reoxygenation-induced damage of cultured hepatocytes, thus suggesting a therapeutic potential in the liver. Therefore, the effects of ANP on hepatic ischemia/reperfusion injury after warm ischemia were studied. METHODS:Livers of male Sprague-Dawley rats subjected to 60 minutes of warm ischemia at 37 degrees C were perfused in the presence or absence of 200 and 20 nmol/L ANP. RESULTS:Sinusoidal lactate dehydrogenase efflux increased to 2000 +/- 264 and 126 +/- 50 mU.min-1.g liver-1 after 1 minute and 60 minutes of reperfusion, but it only increased to 1240 +/- 160 and 22 +/- 16 mU.min-1.g liver-1 in the presence of 200 nmol/L ANP during the preischemic and postischemic perfusion period. The postischemic bile flow (0.67 +/- 0.18 microL.min-1.g liver-1) was significantly improved with 200 nmol/L ANP (0.92 +/- 0.05) and showed a linear correlation to biliary glutathione excretion. In contrast, 20 nmol/L ANP had no protective effects. Administration of 200 nmol/L ANP during the preischemic perfusion period alone (but not after starting reperfusion) markedly preserved postischemic liver function. CONCLUSIONS:Continuous ANP administration or ANP pretreatment alone prevents hepatic ischemia/reperfusion injury, possibly because of influences on intracellular signal transduction processes. The correlation between bile flow and biliary glutathione excretion supports the hypothesis that biliary glutathione transport is one of the osmotic driving forces in postischemic bile formation.
10.1016/s0016-5085(94)94961-1
Ischemic-type biliary complications after orthotopic liver transplantation.
Sanchez-Urdazpal L,Gores G J,Ward E M,Maus T P,Wahlstrom H E,Moore S B,Wiesner R H,Krom R A
Hepatology (Baltimore, Md.)
Nonanastomotic biliary strictures that involve only the biliary tree of the graft occur after orthotopic liver transplantation in patients with hepatic artery thrombosis, chronic ductopenic rejection and ABO blood group incompatibility. This complication may also occur in the absence of these known risk factors. The major focus of our study was to evaluate the risk factors for nonanastomotic biliary stricturing of unknown cause after orthotopic liver transplantation. Results demonstrate that the development of biliary strictures is strongly associated with the duration of cold ischemic storage of allografts in both Euro-Collins solution and University of Wisconsin solution. Results also demonstrate that strictures are not associated with the type of biliary reconstruction, the primary liver disease, cytomegalovirus infection, allograft rejection or the presence of a positive lymphocytotoxic crossmatch. More recently, we have markedly reduced the occurrence of nonanastomotic biliary stricturing by decreasing the ischemia time of our allografts. Thus nonanastomotic biliary strictures appear to be the result of the ischemia/reperfusion-induced tissue injury associated with the harvest and implantation of allografts.
Diagnostic features and clinical outcome of ischemic-type biliary complications after liver transplantation.
Sanchez-Urdazpal L,Gores G J,Ward E M,Maus T P,Buckel E G,Steers J L,Wiesner R H,Krom R A
Hepatology (Baltimore, Md.)
The clinical significance and outcome of nonanastomotic strictures and dilatations involving only the biliary tree of the graft with a radiological appearance of biliary ischemia is unknown. Therefore we analyzed the grafts of 128 patients to evaluate the biochemical, radiological and histological features that prompted the diagnosis of ischemic-type biliary stricture and the clinical outcome of this complication. Ischemic-type biliary strictures were diagnosed in 25 patients (19%). Initial graft function was similar in all patients, whether or not this complication developed. Most ischemic-type biliary strictures occurred between 1 and 4 mo after orthotopic liver transplantation. However, the appearance of ischemic-type biliary stricture in the month after transplantation was predictive for a poor outcome in all six grafts with early onset of ischemic-type biliary strictures. Eighteen patients (72%) were treated with biliary stents and repeated dilatations. Long-term patency was achieved in 88% of these patients. Repeat transplantation was performed in six patients (24%); five survived. Finally, patients with ischemic-type biliary strictures spent more time in the hospital during the first year after orthotopic liver transplantation than did patients without the complication (62 +/- 27 days vs. 37 +/- 20 days; p < or = 0.001). This was due to repeated hospitalizations and a higher incidence of retransplantation. One-year graft survival was lower in patients with ischemic-type biliary strictures than in patients without ischemic-type biliary strictures (69% vs. 88%; p = 0.006). However, 1-yr patient survival was similar in the two groups (91% vs. 89%). In conclusion, early appearance of ischemic-type biliary stricture features is associated with poor graft prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischemia in mice.
Nocito Antonio,Georgiev Panco,Dahm Felix,Jochum Wolfram,Bader Michael,Graf Rolf,Clavien Pierre-Alain
Hepatology (Baltimore, Md.)
UNLABELLED:Hepatic ischemia and reperfusion (I/R) leads to the formation of leukocyte-platelet aggregates. Upon activation, platelets generate reactive oxygen species and release proapoptotic and proinflammatory mediators as well as growth factors. In cold hepatic ischemia, adhesion of platelets to endothelial cells mediates sinusoidal endothelial cell apoptosis. Furthermore, platelet-derived serotonin mediates liver regeneration. We hypothesized that platelets may contribute to reperfusion injury and repair after normothermic hepatic ischemia. The aim of this study was to assess the impact of platelets in normothermic hepatic I/R injury using models of impaired platelet function and immune thrombocytopenia. Inhibition of platelet function in mice was achieved via clopidogrel feeding. Immune thrombocytopenia was induced via intraperitoneal injection of anti-CD41 antibody. Platelet-derived serotonin was investigated using mice lacking tryptophan hydroxylase 1. Mice were subjected to 60 minutes of partial hepatic ischemia and various time points of reperfusion. Hepatic injury was determined via AST and histological analysis of the necrotic area as well as leukocyte infiltration. Liver regeneration was determined via proliferating cell nuclear antigen and Ki67 immunohistochemistry. Neither inhibition of platelet function nor platelet depletion led to a reduction of I/R injury. Liver regeneration and repair were significantly impaired in platelet-depleted animals. Mice lacking peripheral serotonin were deficient in hepatocyte proliferation, but otherwise displayed normal tissue remodeling. CONCLUSION:Platelets have no direct impact on the pathogenesis of normothermic I/R injury. However, they mediate tissue repair and liver regeneration. Furthermore, platelet-derived serotonin is a mediator of hepatocyte proliferation in the postischemic liver, but has no impact on tissue remodeling.
10.1002/hep.21516
Biomarkers to assess graft quality during conventional and machine preservation in liver transplantation.
Verhoeven Cornelia J,Farid Waqar R R,de Jonge Jeroen,Metselaar Herold J,Kazemier Geert,van der Laan Luc J W
Journal of hepatology
A global rising organ shortage necessitates the use of extended criteria donors (ECD) for liver transplantation (LT). However, poor preservation and extensive ischemic injury of ECD grafts have been recognized as important factors associated with primary non-function, early allograft dysfunction, and biliary complications after LT. In order to prevent for these ischemia-related complications, machine perfusion (MP) has gained interest as a technique to optimize preservation of grafts and to provide the opportunity to assess graft quality by screening for extensive ischemic injury. For this purpose, however, objective surrogate biomarkers are required which can be easily determined at time of graft preservation and the various techniques of MP. This review provides an overview and evaluation of biomarkers that have been investigated for the assessment of graft quality and viability testing during different types of MP. Moreover, studies regarding conventional graft preservation by static cold storage (SCS) were screened to identify biomarkers that correlated with either allograft dysfunction or biliary complications after LT and which could potentially be applied as predictive markers during MP. The pros and cons of the different biomaterials that are available for biomarker research during graft preservation are discussed, accompanied with suggestions for future research. Though many studies are currently still in the experimental setting or of low evidence level due to small cohort sizes, the biomarkers presented in this review provide a useful handle to monitor recovery of ECD grafts during clinical MP in the near future.
10.1016/j.jhep.2014.04.031
The influence of cold ischemia time on biliary complications following liver transplantation.
Scotté M,Dousset B,Calmus Y,Conti F,Houssin D,Chapuis Y
Journal of hepatology
Biliary complications are a continuing source of morbidity and mortality following orthotopic liver transplantation. The results of 100 whole-liver allografts performed in 92 adult patients were reviewed to determine whether cold ischemia time and preservation injury influenced both the incidence and type of biliary complications. Mean cold ischemia time was 10.2 +/- 0.5 h (range 3.6-19). Eighteen patients (19.6%) developed 25 biliary complications: there were eight anastomotic leaks, eight anastomotic strictures, six non-anastomotic strictures, two cystic duct mucoceles, and one biliary fistula following T-tube removal. Despite the high rate of reoperative surgery (68%), no death was attributable to biliary complications. Neither cold ischemia time nor early graft function influenced the rate of biliary complications or strictures of either type. Furthermore, an analysis of different factors revealed no predisposing effect of the pre-operative status of the recipient, type of biliary reconstruction, blood requirement, vascular complications, rejection or cytomegalovirus infection on the incidence of biliary complications or strictures. Only chronic rejection could be singled out as a risk factor for non-anastomotic strictures (p = 0.05). These results suggest that prolonged cold ischemia time does not seem to affect the rate or type of biliary complications following orthotopic liver transplantation. In view of these data, there is no clear reason to reconsider prolonged cold ischemia up to 15 h in University of Wisconsin solution, as it has transformed liver transplantation from an emergency operation to a semi-elective procedure and allows longer back-table preparation for graft reduction of splitting.
Preconditioning with death ligands FasL and TNF-alpha protects the cirrhotic mouse liver against ischaemic injury.
Jang J-H,Moritz W,Graf R,Clavien P-A
Gut
BACKGROUND:Ischaemic preconditioning is the preemptive proven strategy to reduce ischaemic injury in the liver, but it can be harmful in the elderly or in patients with liver diseases. Ischaemic preconditioning induces a protective effect via activation of oxidative stress. We hypothesised that Fas ligand and tumour necrosis factor alpha can induce a similar response. Therefore, we tested if death ligands could mimic ischaemic preconditioning. METHODS:Ischaemia was maintained for 60 min in cirrhotic mice. Death ligands were given 40 min before ischaemia. Ischaemic injury was assessed by histology and biochemical assays. To elucidate the mechanism, we used zinc protophorphyrin, an inhibitor of haem oxygenase-1 (HO-1), and gadolinium chloride, an inhibitor of Kupffer cells. RESULTS:Compared with the control group, death ligand preconditioning strongly reduced all markers of injury: serum transaminase levels, necrosis and apoptosis. Preconditioning caused an upregulation of HO-1, predominantly in macrophages. When zinc protophorphyrin or gadolinium chloride was applied prior to preconditioning, the beneficial effect of preconditioning was lost. CONCLUSION:These results demonstrate that ischaemic preconditioning can be replaced by death ligand preconditioning in the cirrhotic liver to prevent ischaemic injury. The protective mechanism depends on HO-1 induction in macrophages. These results open doors for novel hepato-protective strategies in liver surgery and transplantation.
10.1136/gut.2007.137703
Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver.
Koeppel T A,Trauner M,Mennone A,Arrese M,Rios-Velez L,Boyer J L
Journal of hepatology
BACKGROUND/AIMS:Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS:We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS:Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS:We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.
Cholestasis protects the liver from ischaemic injury and post-ischaemic inflammation in the mouse.
Georgiev P,Navarini A A,Eloranta J J,Lang K S,Kullak-Ublick G A,Nocito A,Dahm F,Jochum W,Graf R,Clavien P-A
Gut
BACKGROUND AND HYPOTHESIS:Cholestasis is associated with high morbidity and mortality in patients undergoing major liver surgery, but the mechanisms responsible remain elusive. Increased ischaemic liver injury and inflammation may contribute to the poor outcome. METHODS:Common bile duct ligation (biliary obstruction with hyperbilirubinaemia) or selective ligation of the left hepatic duct (biliary obstruction without hyperbilirubinaemia) was performed in C57BL/6 mice before 1 h of hepatic ischaemia and 1, 4 or 24 h of reperfusion. Infection with the intracellular hepatic pathogen Listeria monocytogenes for 12 and 48 h was used to study ischaemia-independent hepatic inflammation. RESULTS:Cholestatic mice showed considerable protection from ischaemic liver injury as determined by transaminase release, histological liver injury and neutrophil infiltration. In cholestatic mice, reduced injury correlated with a failure to activate nuclear factor kappaB (NFkappaB) and tumour necrosis factor alpha (TNFalpha) mRNA synthesis, two key mediators of post-ischaemic liver inflammation. After selective bile duct ligation, both the ligated and the non-ligated lobes showed blocked activation of NFkappaB as well as reduced induction of TNFalpha mRNA synthesis and neutrophil infiltration. By contrast, infection with L monocytogenes showed comparable activation of NFkappaB and hepatic recruitment of neutrophils 12 h after infection. CONCLUSION:Cholestasis does not increase but rather dramatically protects the liver from ischaemic injury and inflammation. This effect is mediated by a systemic factor, but not bilirubin, and is associated with a preserved capacity to trigger an inflammatory response to other stimuli such as a bacterial pathogen.
10.1136/gut.2006.097170
MMP-9 deficiency shelters endothelial PECAM-1 expression and enhances regeneration of steatotic livers after ischemia and reperfusion injury.
Kato Hiroyuki,Kuriyama Naohisa,Duarte Sergio,Clavien Pierre-Alain,Busuttil Ronald W,Coito Ana J
Journal of hepatology
BACKGROUND & AIMS:Organ shortage has led to the use of steatotic livers in transplantation, despite their elevated susceptibility to ischemia/reperfusion injury (IRI). Matrix metalloproteinase-9 (MMP-9), an inducible gelatinase, is emerging as a central mediator of leukocyte traffic into inflamed tissues. However, its role in steatotic hepatic IRI has yet to be demonstrated. METHODS:We examined the function of MMP-9 in mice fed with a high-fat diet (HFD), which developed approximately 50% hepatic steatosis, predominantly macrovesicular, prior to partial hepatic IRI. RESULTS:The inability of MMP-9(-/-) deficient steatotic mice to express MMP-9 significantly protected these mice from liver IRI. Compared to fatty controls, MMP-9(-/-) steatotic livers showed significantly reduced leukocyte infiltration, proinflammatory cytokine expression, and liver necrosis. Loss of MMP-9 activity preserved platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, a modulator of vascular integrity at the endothelial cell-cell junctions in steatotic livers after IRI. Using in vitro approaches, we show that targeted inhibition of MMP-9 sheltered the extracellular portion of PECAM-1 from proteolytic processing, and disrupted leukocyte migration across this junctional molecule. Moreover, the evaluation of distinct parameters of regeneration, proliferating cell nuclear antigen (PCNA) and histone H3 phosphorylation (pH3), provided evidence that hepatocyte progression into S phase and mitosis was notably enhanced in MMP-9(-/-) steatotic livers after IRI. CONCLUSIONS:MMP-9 activity disrupts vascular integrity at least partially through a PECAM-1 dependent mechanism and interferes with regeneration of steatotic livers after IRI. Our novel findings establish MMP-9 as an important mediator of steatotic liver IRI.
10.1016/j.jhep.2013.12.022
Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation.
Brunner Stefan M,Junger Henrik,Ruemmele Petra,Schnitzbauer Andreas A,Doenecke Axel,Kirchner Gabriele I,Farkas Stefan A,Loss Martin,Scherer Marcus N,Schlitt Hans J,Fichtner-Feigl Stefan
Journal of hepatology
BACKGROUND & AIMS:The aim of this study was to examine the development of biliary epithelial damage between organ retrieval and transplantation and its clinical relevance for patients. METHODS:Common bile duct samples during donor hepatectomy, after cold storage, and after reperfusion were compared to healthy controls by hematoxylin and eosin (H&E) staining and immunofluorescence for tight junction protein 1 and Claudin-1. A bile duct damage score to quantify biliary epithelial injury was developed and correlated with recipient and donor data and patient outcome. RESULTS:Control (N=16) and donor hepatectomy bile ducts (N=10) showed regular epithelial morphology and tight junction architecture. After cold storage (N=37; p=0.0119), and even more after reperfusion (N=62; p=0.0002), epithelial damage, as quantified by the bile duct damage score, was markedly increased, and both tight junction proteins were detected with inappropriate morphology. Patients with major bile duct damage after cold storage had a significantly increased risk of biliary complications (relative risk 18.75; p<0.0001) and graft loss (p=0.0004). CONCLUSIONS:In many cases, the common bile duct epithelium shows considerable damage after cold ischemia with further damage occurring after reperfusion. The extent of epithelial damage can be quantified by our newly developed bile duct damage score and is a prognostic parameter for biliary complications and graft loss. Possibly, in an intraoperative histological examination, this bile duct damage score may influence decision-making in transplantation surgery.
10.1016/j.jhep.2012.12.022
Machine perfusion in liver transplantation as a tool to prevent non-anastomotic biliary strictures: Rationale, current evidence and future directions.
Weeder Pepijn D,van Rijn Rianne,Porte Robert J
Journal of hepatology
The high incidence of non-anastomotic biliary strictures (NAS) after transplantation of livers from extended criteria donors is currently a major barrier to widespread use of these organs. This review provides an update on the most recent advances in the understanding of the etiology of NAS. These new insights give reason to believe that machine perfusion can reduce the incidence of NAS after transplantation by providing more protective effects on the biliary tree during preservation of the donor liver. An overview is presented regarding the different endpoints that have been used for assessment of biliary injury and function before and after transplantation, emphasizing on methods used during machine perfusion. The wide spectrum of different approaches to machine perfusion is discussed, including the many different combinations of techniques, temperatures and perfusates at varying time points. In addition, the current understanding of the effect of machine perfusion in relation to biliary injury is reviewed. Finally, we explore directions for future research such as the application of (pharmacological) strategies during machine perfusion to further improve preservation. We stress the great potential of machine perfusion to possibly expand the donor pool by reducing the incidence of NAS in extended criteria organs.
10.1016/j.jhep.2015.03.008
Hypothermic oxygenated perfusion (HOPE) protects from biliary injury in a rodent model of DCD liver transplantation.
Schlegel Andrea,Graf Rolf,Clavien Pierre-Alain,Dutkowski Philipp
Journal of hepatology
BACKGROUND & AIMS:The use of livers from donors after cardiac arrest (DCD) is increasing in many countries to overcome organ shortage. Due to additional warm ischemia before preservation, those grafts are at higher risk of failure and bile duct injury. Several competing rescue strategies by machine perfusion techniques have been developed with, however, unclear effects on biliary injury. We analyze the impact of an end-ischemic Hypothermic Oxygenated PErfusion (HOPE) approach applied only through the portal vein for 1h before graft implantation. METHODS:Rat livers were subjected to 30-min in situ warm ischemia, followed by subsequent 4-h cold storage, mimicking DCD-organ procurement and conventional organ transport. Livers in the HOPE group underwent also passive cold storage for 4h, but were subsequently machine perfused for 1h before implantation. Outcome was tested by liver transplantation (LT) at 12h after implantation (n=10 each group) and after 4 weeks (n=10 each group), focusing on early reperfusion injury, immune response, and later intrahepatic biliary injury. RESULTS:All animals survived after LT. However, reperfusion injury was significantly decreased by HOPE treatment as tested by hepatocyte injury, Kupffer cell activation, and endothelial cell activation. Recipients receiving non-perfused DCD livers disclosed less body weight gain, increased bilirubin, and severe intrahepatic biliary fibrosis. In contrast, HOPE treated DCD livers were protected from biliary injury, as detected by cholestasis parameter and histology. CONCLUSIONS:We demonstrate in a DCD liver transplant model that end-ischemic hypothermic oxygenated perfusion is a powerful strategy for protection against biliary injury.
10.1016/j.jhep.2013.06.022
MicroRNA profiles in graft preservation solution are predictive of ischemic-type biliary lesions after liver transplantation.
Verhoeven Cornelia J,Farid Waqar R R,de Ruiter Petra E,Hansen Bettina E,Roest Henk P,de Jonge Jeroen,Kwekkeboom Jaap,Metselaar Herold J,Tilanus Hugo W,Kazemier Geert,van der Laan Luc J W
Journal of hepatology
BACKGROUND & AIMS:Ischemic-type biliary lesions (ITBL) are the second most common cause of graft loss after liver transplantation. Though the exact pathophysiology of ITBL is unknown, bile duct injury during graft preservation is considered to be a major cause. Here we investigated whether the release of cholangiocyte-derived microRNAs (CDmiRs) during graft preservation is predictive of the development of ITBL after liver transplantation. METHODS:Graft preservation solutions (perfusates) and paired liver biopsies collected at the end of cold ischemia were analysed by RT-qPCR for CDmiR-30e, CDmiR-222, and CDmiR-296 and hepatocyte-derived miRNAs (HDmiRs) HDmiR-122 and HDmiR-148a. MicroRNAs in perfusates were evaluated on their stability by incubation and fractionation experiments. MicroRNA profiles in perfusates from grafts that developed ITBL (n=20) and grafts without biliary strictures (n=37) were compared. RESULTS:MicroRNAs in perfusates were proven to be stable and protected against degradation by interacting proteins. Ratios between HDmiRs/CDmiRs were significantly higher in perfusates obtained from grafts that developed ITBL (p<0.01) and were identified as an independent risk factor by multivariate analysis (p<0.01, HR: 6.89). The discriminative power of HDmiRs/CDmiRs in perfusates was validated by analysis of separate brain death- (DBD) and cardiac death donors (DBD; p ≤ 0.016) and was superior to expression in liver biopsies (C=0.77 in perfusates vs. C<0.50 in biopsies). CONCLUSIONS:This study demonstrates that differential release of CDmiRs during graft preservation is predictive of the development of ITBL after liver transplantation. This provides new evidence for the link between graft-related bile duct injury and the risk for later development of ITBL.
10.1016/j.jhep.2013.07.034
Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.
op den Dries Sanna,Westerkamp Andrie C,Karimian Negin,Gouw Annette S H,Bruinsma Bote G,Markmann James F,Lisman Ton,Yeh Heidi,Uygun Korkut,Martins Paulo N,Porte Robert J
Journal of hepatology
BACKGROUND & AIMS:The peribiliary glands of large bile ducts have been identified as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. We aimed to determine whether injury to the peribiliary glands of donor livers is a risk factor for development of non-anastomotic biliary strictures (NAS) after liver transplantation. METHODS:In 128 liver transplant procedures, biopsies were taken from the donor bile duct and injury was assessed using an established histological grading system. Histological severity of injury was subsequently compared in liver grafts that later developed biliary structures vs. uncomplicated liver grafts. RESULTS:Luminal biliary epithelial loss >50% was observed in 91.8% of the grafts before transplantation, yet NAS occurred in only 16.4%. Periluminal peribiliary glands were more severely injured than deep peribiliary glands located near the fibromuscular layer (>50% loss in 56.9% vs. 17.5%, respectively; p<0.001). Injury of deep peribiliary glands was more prevalent and more severe in livers that later developed NAS, compared to grafts without NAS (>50% loss in 50.0% vs. 9.8%, respectively; p=0.004). In parallel, injury of the peribiliary vascular plexus was more severe in livers that developed NAS, compared to grafts without NAS (>50% vascular changes in 57.1% vs. 20.3%; p=0.006). CONCLUSION:Injury of peribiliary glands and vascular plexus before transplantation is strongly associated with the occurrence of biliary strictures after transplantation. This suggests that insufficient regeneration due to loss of peribiliary glands or impaired blood supply may explain the development of biliary strictures.
10.1016/j.jhep.2014.02.010